1. Volume 22, Issue 7, Pages 985-995 (July 2014)
Spontaneous Self-Assembly of Engineered Armadillo Repeat Protein Fragments into a Folded Structure 
Randall P. Watson, Martin T. Christen, Christina Ewald, Fabian Bumbak, Christian Reichen, Maja Mihajlovic, Elena Schmidt, Peter Güntert, Amedeo Caflisch, Andreas Plückthun, Oliver Zerbe 
Structure 
Volume 22, Issue 7, Pages (July 2014)
DOI: /j.str
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2. Structure 2014 22, 985-995DOI: (10.1016/j.str.2014.05.002)
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3. Figure 1 [15N, 1H]-HSQC Spectra
(A) 15N-Labeled MA in the absence of YM2.
(B) 15N-Labeled YM2 in the absence of MA.
(C) Expansion of the spectrum of 15N-labeled MA in complex with unlabeled YM2 (dotted lines) at a 1:1.2 molar ratio, superimposed with the spectrum of 15N-labeled MA in the absence of YM2 (solid lines).
(D) 15N-Labeled YM2 complexed with unlabeled MA at 1:1.2 molar ratio. A schematic drawing of the modular nature of YM3A and the fragments is depicted in the inset in (D).
All spectra were recorded on a 700 MHz spectrometer at 307 K in 50 mM sodium phosphate buffer with 150 mM NaCl, 2% glycerol, 0.02% NaN3, and 10% D2O (pH 7.4).
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4. Figure 2 ITC Isotherm and Curve Fitting for the YM2:MA Interaction
(A) ITC isotherm.
(B) Curve fitting.
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5. Figure 3 Analytical Size Exclusion Analysis
YM2 behaves as a monomer at low concentrations and shows oligomerization at higher concentrations (top right). MA elutes as one monomeric peak at all tested concentrations (top left). The complex of YM2 and MA elutes as one monomeric peak (bottom left) with the same elution volume as full-length YM3A (bottom right) at all tested concentrations. The arrows indicate positions (from left to right) of exclusion volumes corresponding to the void volume and proteins with molecular weights of 44.3, 25.0, and 13.7 kDa.
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6. Figure 4 NMR Structures of MA
(A) Twenty lowest energy conformers of uncomplexed MA.
(B) Twenty lowest energy conformers of MA in complex with unlabeled YM2.
In both cases, the conformer bundles are superimposed with the corresponding region from the crystal structure (PDB accession number 4DBA, red) of the entire protein, YM3A. The location of the C terminus of MA is indicated.
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7. Figure 5
Distance Constraints Mapped on the Closest-to-Average NMR Conformers of the Armadillo Complex
The complexed YM2 (green) and MA (cyan) fragments are visualized as cartoons; residues 1 to 34 are omitted for clarity. Upper-limit distance constraints are shown for intramolecular restraints (A) (yellow) and interfacial restraints (B) (red).
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8. Figure 6 3D Structures of the YM2:MA Complex
The polypeptide backbone is shown in stick representation. NMR structures are colored by fragment: YM2 (green) and MA (cyan). Note that the two fragments are not covalently linked. Locations of termini are indicated by letters; the unrestrained residues 1 to 34 are omitted for clarity.
(A) Superposition of 20 NMR structures.
(B) Closest-to-average NMR structure superimposed onto the crystal structure of full-length YM3A (PDB accession number 4DBA, red).
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9. Figure 7
Interface between the YM2 and MA Fragments in an “Open Book” View
The Connolly contact surface is colored by amino acid charge: nonpolar side chains are depicted in yellow, acidic in red, basic in blue, and polar uncharged in green. For this visualization, the closest-to-average NMR structure of the YM2:MA complex was separated at the interface and the two fragments splayed as indicated.
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10. Figure 8
MD Simulations
(A) Structural stability of the YM2:MA complex analyzed by MD simulations through comparison with the full-length protein. The time series of the rmsd from the crystal structure (PDB accession number 4DBA) were calculated for the Cα atoms of repeats M1M2M3A (top) and M2M3A (middle) to the crystal structure. Note that only in the xtal run is M2 covalently linked to M3.
(B) Time series of the distance between the carboxyl C atom of Gly115 and the amino N atom of Gly116 termini along the MD simulations, which started from the lowest energy NMR structure of runs NMR1 and NMR3 and the split-xtal runs.
Structure  , DOI: ( /j.str )
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